Linear free energy relationships in cAMP-dependent protein kinase reactions with synthetic substrates

Abstract The quantitative structure-activity relationships for cAMP-dependent protein kinase reactions with synthetic substrates were used to analyze the mechanism of peptide phosphorylation and specificity of the enzyme. It was shown that in most cases the variation in peptide structure affected the binding affinity of substrate but had only a weak effect on the observed rate of the phosphorylation reaction. Moreover, the intrinsic reactivity of the phosphorylatable OH-group has no effect on the apparent rate of the catalytic step, pointing to the fact that the chemical act of peptide phosphorylation is not the rate-limiting step of the overall process. Introduction of a proline residue after the phosphorylatable amino acid changes the specificity pattern of the enzyme. Variation in the structure of the latter type of substrates alters the observed rate of phosphorylation of peptides while it has almost no effect on their binding affinity. The influence of structure of amino acids on the binding effectiveness of peptides was analyzed by taking into account hydrophobicity and steric properties of their sidechains, quantified by the π-constants and steric constants E s o .